Light-mediated polymerase chain reaction amplification and product detection system and methods of use

Abstract

A PCR amplification and product detection system is disclosed. The system utilizes a uniform and direct photonic heating subsystem to mediate reaction-by-reaction, high-throughput PCR amplification detectable by a fluorescence detection subsystem. Reaction-by-reaction temperature monitoring for dynamic feedback heat regulation is also disclosed. Also disclosed are methods for using the same.

That which is claimed: 
     
       1. A polymerase chain reaction (PCR) amplification and detection apparatus, comprising:
 a) an assembly subsystem comprising:
 i) a plurality of vessels in the form of a continuous polymer strip, each such vessel having a volume of 0.2 to 20 μl and a vessel diameter of 0.5 to 4 mm; 
 ii) said plurality of vessels each comprising an aqueous oil matrix, each aqueous oil matrix comprising:
 1) an aqueous reaction mix comprising a polynucleotide sample and reagents, the reagents comprising a first reagent capable of excitation by a fluorescence excitation light having a first spectral wavelength when the first reagent hybridizes to the polynucleotide sample; and 
 2) one or two non-miscible oils selected from the group consisting of an encapsulation oil, a carrier oil, and both an encapsulation oil and a carrier oil; 
 wherein components of the aqueous reaction mix do not mix with the one or two non-miscible oils; 
 
 
 b) a plurality of heating positions, temperature monitoring positions, and PCR product detection positions defining a plurality of alternating first vessels stations and second vessels stations where the plurality of vessels may be stationed, and wherein each of the first vessels stations comprises a heating position, and each of the second vessels stations comprises a PCR product detection position; 
 c) a reaction-by-reaction, light-driven photonic heating subsystem comprising a plurality of electromagnetic radiation sources, wherein, when the plurality of vessels are in the heating position, each vessel is in optical communication with an electromagnetic radiation source, and the electromagnetic radiation source emits electromagnetic radiation to that vessel; 
 d) a reaction-by-reaction temperature monitoring subsystem comprising a plurality of thermal detection devices, wherein each vessel corresponds to a thermal detection device the thermal detection device is configured to provide a measuring signal dependent on the temperature of the aqueous oil matrix contained in the vessel; 
 e) a microcontroller temperature feedback and light source control subsystem communicatively connected to both the photonic heating subsystem and the temperature monitoring subsystem, wherein the microcontroller temperature feedback and light source control subsystem is configured to regulate an energy input required for controlling an output and a duration of an electromagnetic energy emitted by each electromagnetic radiation source through a cycle of reaction temperatures; 
 f) a fluorescence detection subsystem comprising:
 i) one or more fluorescence excitation light sources; 
 ii) one or more fluorescence emission light sensing devices; 
 iii) a plurality of first optical members in optical communication with the one or more fluorescence excitation light sources, wherein, when the vessels are in the PCR detection positions, each first optical member is configured to provide an optical path for fluorescence excitation light having the first spectral wavelength from the one or more fluorescence excitation light sources to one of said vessels containing the aqueous oil matrix, and wherein each first optical member is further configured to provide an optical path for fluorescence emission light from the aqueous reaction mix to the one or more fluorescence emission light sensing devices; and 
 iv) an active or passive cooling mechanism at the PCR product detection positions whereby each of the vessels in the PCR product detection positions are cooled; and 
 
 g) a mechanical and electronic control system communicatively connected to a moveable belt and to the assembly subsystem, wherein the mechanical and electronic control system causes the assembly subsystem to assemble the aqueous oil matrices in the plurality of vessels, and wherein the mechanical and electronic control system causes the moveable belt to move the plurality of vessels with the aqueous oil matrix in a step-by-step movement through the assembly subsystem in order to align the plurality of vessels with the aqueous oil matrix with the plurality of alternating first vessels stations and second vessels stations. 
 
     
     
       2. The apparatus of  claim 1 , wherein each of the first vessels stations comprise a heating position and a temperature monitoring position, and wherein each of the second vessels stations comprise a PCR product detection position. 
     
     
       3. The apparatus of  claim 1 , wherein each of the first vessels stations comprise a heating position, and wherein each of the second vessel stations comprise a PCR product detection position and a temperature monitoring position. 
     
     
       4. The apparatus of  claim 1 , wherein each electromagnetic radiation source is configured to uniformly heat the volume of the aqueous reaction mix in each corresponding vessel when the vessel is in the heating position through the cycle of reaction temperatures comprising: (i) an annealing temperature in the range from about 50° C. to about 65° C.; (ii) an elongation temperature in the range from about 65° C. to about 75° C.; and (iii) a denaturation temperature in the range from about 90° C. to about 99° C.; and wherein the active or passive cooling mechanism is configured to cool each of the vessels to a temperature range from about 55° C. to about 65° C. when the vessel is in the PCR product detection position. 
     
     
       5. The apparatus of  claim 4 , wherein the aqueous reaction matrix is heated through the cycle of temperatures (i), (ii), and (iii), in less than or equal to 20 seconds, and wherein the volume of the aqueous reaction mix portion of the aqueous reaction matrix is less than or equal to 10 μL. 
     
     
       6. The apparatus of  claim 5 , wherein the aqueous reaction matrix is heated through the cycle of temperatures (i), (ii), and (iii), in less than or equal to 15 seconds, and wherein the volume of the aqueous reaction mix portion of the aqueous reaction matrix is less than or equal to 10 μL. 
     
     
       7. The apparatus of  claim 6 , wherein the aqueous reaction matrix is heated through the cycle of temperatures (i), (ii), and (iii), in less than or equal to 10 seconds, and wherein the volume of the aqueous reaction mix portion of the aqueous reaction matrix is less than or equal to 10 μL. 
     
     
       8. The apparatus of  claim 6 , wherein the cycle of reaction temperatures in (i), (ii), and (iii) is repeated for an additional 1 to 60 cycles. 
     
     
       9. The apparatus of  claim 1 , wherein the fluorescence detection subsystem further comprises:
 v) a first excitation filter which receives light from the one or more fluorescence excitation light sources and allows passage of the fluorescence excitation light having the first spectral wavelength; 
 vi) a first emission filter for allowing transmission therethrough of fluorescence emission light to the one or more fluorescence emission light sensing devices from the aqueous reaction mix in response to the fluorescence excitation light having the first spectral wavelength and for substantially blocking transmission of wavelengths other than the wavelengths of the emitted light; or 
 vii) both v) and vi). 
 
     
     
       10. The apparatus of  claim 1 , wherein each first optical member is further configured to provide an optical path for fluorescence excitation light having a second spectral wavelength from the one or more fluorescence excitation light sources to one of said vessels containing the aqueous oil matrix when the vessel is in a PCR product detection position, wherein the volume of the aqueous reaction mix comprises a second reagent capable of excitation by the fluorescence excitation light having the second spectral wavelength when the second reagent hybridizes to the DNA sample, and wherein each first optical member is further configured to provide an optical path for fluorescence emission light from the aqueous reaction mix to the one or more fluorescence emission light sensing devices. 
     
     
       11. The apparatus of  claim 10 , wherein the first reagent, the second reagent, or both the first reagent and the second reagent comprises a nucleic acid probe covalently linked to a fluorophore. 
     
     
       12. The apparatus of  claim 1 , wherein the one or more fluorescence emission light sensing devices is a charged-coupled device (CCD) camera, complimentary metal-oxide semiconductor (CMOS) camera, sensor array, or a combination thereof. 
     
     
       13. The apparatus of  claim 1 , wherein each vessel comprises the aqueous reaction mix, the carrier oil, and the encapsulation oil, wherein the carrier oil is non-miscible with both the encapsulation oil and the components of the aqueous reaction mix. 
     
     
       14. The apparatus of  claim 13 , wherein the carrier oil has a density ranging from about 1,200 kg/m 3  to about 2,000 kg/m 3 , the encapsulation oil has a density ranging from about 700 kg/m 3  to about 990 kg/m 3 , and the aqueous reaction mix has a density ranging from about 900 kg/m 3  to about 1,200 kg/m 3 . 
     
     
       15. The apparatus of  claim 1 , wherein the electromagnetic radiation sources are laser diodes emitting infrared light having a spectral wavelength in the range from about 1,300 nm to about 2,200 nm. 
     
     
       16. The apparatus of  claim 1 , wherein the electromagnetic radiation sources are laser diodes emitting infrared light having a spectral wavelength in the range from about 1,300 nm to about 1,500 nm. 
     
     
       17. The apparatus of  claim 1 , further comprising one or more hot start heating positions prior to the plurality of heating positions. 
     
     
       18. The apparatus of  claim 1 , wherein the photonic heating subsystem further comprises a lens that focuses the electromagnetic radiation of the photonic heating subsystem onto a metallic film disposed between the bottom of the vessel and the oil. 
     
     
       19. The apparatus of  claim 1 , wherein the vessels are uncovered. 
     
     
       20. The apparatus of  claim 19 , wherein the fluorescence detection subsystem is positioned in optical communication with the top of the vessel. 
     
     
       21. The apparatus of  claim 20 , wherein the photonic heating subsystem is positioned above or below the vessel.